Current efforts have been devoted to examining the nature of the allo-determinants recognized by cloned T cell populations. To examine this question, H-2 structural mutants have been isolated from a somatic cell line by mutagenesis and immuno-selection using monoclonal anti-H-2 antibodies. Examination of alloantigen-specific CTL clones on these mutants suggest that the majority of CTL clones recognize determinants different from those which elicit antibody production. Analysis of the in vitro-derived mutants has shown that new determinants are created by the mAb immunoselection procedure which can be recognized by cytotoxic T cells. The new allodeterminants expressed on the in vitro-derived CTL can function as transplantation antigens in vivo and appear linked to a single amino acid substitution. In addition, the regions of the MHC molecule involved in CTL recognition were studied using L cells transfected with H-2 genes constructed by shuffling exons between the H-2Ld and H-2Dd genes. The findings suggested that unlike mAbs which can recognize individual epitopes on different domains, a majority of the CTL clones recognize determinants influenced by the interaction of the two external domains. In one instance, CTL can recognize a hybrid Dd/Ld molecule (T9-10-3). However, the CTL employ a restricted TcR VBeta chain family. Other altered MHC class I genes have also been examined including L cells which have been transfected with truncated Ld and Dd genes and express only the Alpha3/TM portion of the molecule. The finding demonstrated that CTL can be generated against truncated MHC gene products. Finally, L cells transfected with hybrid MHC genes between mouse and human class I genes have also been examined. In some instances, CTL clones which recognize native Kb-transfected L cells do not recognize MHC hybrid molecules that have human alpha3 domain. Thus, the specificity of CTL although predomently determined by the Alpha1 + Alpha2 domains is critically influenced by alpha 3.